Abstract
Interstitial deletion or loss of chromosome 5 is a frequent chromosomal abnormality in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Previous studies uncovered a 1.5–2Mb region on 5q31.1 to be a common deleted region (CDR) in those diseases. It is believed that there exists one or more of critical tumor suppressor gene(s) in this CDR. Mutation analysis, however, failed to identify target gene(s). With a hypothesis that epigenetic silencing may play a significant role as a “second hit” in MDS/AML, we performed a comprehensive methylation analysis of genes in this CDR on 5q31.1. Among 25 genes that are located in this region (SPOCK-AF116688, covering CDR determined by Horrigan et al. Blood 2000 and Fang et al. Genomics 2000), 14 genes had promoter regions in CpG islands that were therefore susceptible to methylation dependent silencing. Methylation status of multiple CpG sites in promoter regions of these those 14 genes (SPOCK, HNRPA1, PKD2L2, C5orf5, KIF20A, GFRA3, CDC25C, C5orf6, JMJD1B, C5orf19, EGR1, ETF1, HSPA9B, CTNNA1, SIL1) was analyzed using bisulfite-PCR followed by pyrosequencing. Analyzed samples were 13 leukemic cell lines as well as peripheral blood mononuclear cells from 5 patients with AML with 5q deletion (5qAML), 9 AML without 5q deletion (non-5qAML), 17 MDS with 5q deletion (5qMDS), 9 MDS without 5q deletion (non-5qMDS) and 10 individuals without leukemia. Significant hypermethylation (>15% by pyrosequencing) was observed in 5 genes in patient samples (SPOCK, PKD2L2, GFRA3, HSPA9B, and ETF1, see Table 1) and 3 other genes in cell lines only (CDC25C, C5orf19, CTNNA1). In 5qMDS, the most frequently methylated genes were ETF1 (5/17), GFRA3 (4/17), and HSPA9B (11/17), but HSPA9B also showed methylation in normal blood. We then evaluated the expression levels of 5 genes that showed significant methylation in patients’ samples using quantitative RT-PCR. SPOCK, PKD2L2, GFRA3 were not expressed in normal PBMC. HSPA9B expression levels did not correlate with the degree of methylation. On the other hand, ETF1 expression was inversely correlated with the degree of methylation. ETF1 is a eukaryotic translation termination factor, which plays a role in recognizing stop codon in mRNA and terminates translation. Based on specific hypermethylation and silencing in 5qMDS, we propose ETF1 as a candidate tumor suppressor gene in the 5q31.1 region. Functional analysis of ETF1 in leukemic cells is underway.
Frequency of promoter CpG sites methylation Samples N SPOCK PKD2L2 GFRA3 HSPA9B ETF1 Cell lines 13 62% 46% 77% 38% 0% 5qAML 5 0% 0% 0% 20% 0% Non-5qAML 9 33% 0% 0% 0% 0% 5qMDS 17 6% 6% 24% 65% 29% Non-5qMDS 9 11% 11% 33% 33% 11% Normal 10 0% 0% 0% 90% 0%
Expression of L-myc and N-myc proto-oncogenes in human leukemias and leukemia cell lines